| Part I The influence of blood circulation components in JTXKG on glycolipid metabolism of HFD introduced obesity C57BL/6J miceObjectiveThis research adopted the high fat diet introduced C57BL/6J obese mice,through the detection of lipid metabolism and lipid metabolism related indexes in serum and adipose tissue of C57BL/6J mice,to observe the effect of JTXKG HFD on the metabolism of glucose and lipid in C57BL/6J mice obesity and obesity-related glucose and lipid metabolism;explore the possible mechanism of JTXKG blood circulation components in regulating adipose tissue function and improving the metabolism of glucose and lipid in the adipose tissue of obese C57BL/6J adipose tissue and the key transcription factors of adipogenic differentiation.MethodsEighty 6-weeks old C57BL/6J male mice divided into normal control group(n = 10)and high fat group(n = 60),the mice in the normal control group were fed with normal diet and the high fat diet group fed the high fat diet.After 12 weeks,20%of the mean body weight of the normal diet group was the criteria for obesity model,The obese mice were randomly divided into model group,metformin group,salvianolic acid group B and curcumin group(n = 11).Each group were given oral administration by 10g.BW/0.1ml volume,salvianolic acid B dose:100mg/kg.BW/day;Curcumin dose:50mg/kg.BW/day;Metformin administered at a dose of 75 mg/kg body weight,model group and normal group were given the same amount of deionized water.Total administration for 8 weeks.Body weight,24h intake,FBG were measured at the same time each week.Body fat content and oral glucose tolerance were measured at the 4th and 8th week.The levels of FBG,TG,TC,HDL,LDL,FFA and liver function were measured after the end of the experiment.HE staining observation of adipose tissue The mRNA expression of adiponectin,ATGL,HSL,p3-AR,CEBP a,PPARa,PPARy and SREBP-lc in adipose splitting and adipogenic differentiation genes were detected by RT-PCR.The protein expression of ATGL,HSL,β3-AR,CEBP α,PPARα,PPARγ and SREBP-1 in adipose tissue were detected by Western Blot.ResultsFor the weight change,we found that there were 29(48.3%)mice in the HFD group at 8 weeks of high fat diet.At 12 weeks,47 obese models were successful in HFD group,and the rate of molding was 78.3%.After 8 weeks of treatment,the body weight of the mice in the model group increased by 15.71 g on average;the body weight of the mice in the normal group increased by 8.315g;the body weight of the mice in the salvianolic acid group B increased by 0.987g;the weight of the curcumin group increased by 2.07g;The weight of the positive drug metformin group was 5.092g.Compared with the model group,the weight gain of the treatment group was significantly lower than that of the model group(P<0.01 in the metformin group,P<0.01)and the curcumin group(P<0.05).(P<0.05).Compared with the model group,the body fat levels of the mice in the model group were significantly higher than those in the normal group(P<0.05).Compared with the model group,the rate of systemic fat was significantly lower than that of the control group(P<0.05),but the difference was statistically significant.The body fat rate of the mice treated with salvianolic acid B and curcumin group was higher than that of the model group Decreased,but no statistically significant difference.At the 8th week of treatment,the body fat levels of-the mice in the treatment group were significantly lower than those in the model group(P<0.01 for salvianolic acid B,curcumin group and P<0.001 for metformin group).The food intake of each group showed an increasing trend,and salvianolic acid B and curcumin did not affect the food intake of mice under the dose of the drug used in this study.Compared with the model group,the blood glucose began to decrease after the second week of treatment in the treatment group,and the blood glucose level decreased at the 5th week,and reached the lowest peak after treatment.Then the salvianolic acid group B At the 7th and 8th week,the blood glucose level of each treatment group was significantly lower than that of the model group(P<0.01).At 4 weeks,OGTT results showed that the area under the curve(AUC)was lower in the treatment group than in the model group(P<0.01),especially in the metformin group.At 8 weeks,the area under the OGTT curve showed that the AUC of the treatment group was smaller than that of the model group(P<0.01).The levels of serum TG,TC,LDL-C and FFA in obese mice were decreased after 8 weeks of treatment(P<0.01,P<0.05),while serum HDL-C levels were significantly higher than those in model mice(P<0.01).After 8 weeks of treatment,the levels of serum ALT and AST in salvianolic acid B and curcumin group were lower than those in model group(P<0.01).The expression of Adiponectin,ATGL and β3-AR mRNA was significantly up-regulated by metformin compared with the model group(P<0.05),and the salbutamol group B was significantly up-regulated in the brown adipose tissue(BAT)The expression of HSL mRNA(P<0.05).The expression of β3-AR mRNA was up-regulated in curcumin group(P<0.05).The expression of C/EBPα and PPARγ mRNA was up-regulated by salvianolic acid B and metformin in the expression of adipogenic transcription factor(P<0.05),but there was no significant difference(P<0.05)Gt;0.05).There was no significant effect of PPARα and SREBP-1 mRNA expression in BAT.The mRNA expression of Adiponectin,ATGL and β3-AR in the metformin group was significantly higher than that in the model group(P<0.05),and the expression of HSL in the curcumin group was significantly higher than that in the model group(P<0.05),MRNA expression of β3-AR(P<0.05).Salvianolic acid group B had a tendency to up-regulate ATGL,HSL and β3-AR mRNA,but there was no significant difference(P>0.05).Compared with the model group,the mRNA expression of C/EBPα was up-regulated in metformin group and salvianolic acid group B(P<0.05),and the expression of C/EBPα mRNA was significantly higher in the treatment group(P<0.05)Upregulation of PPARγ mRNA expression(salvianolic acid B,curcumin P<0.05;metformin P<0.01).The mRNA expression of PPARα and SREBP-1 in WAT was not significantly different from that in model group.The expression of ATGL protein in the treatment group was significantly higher than that in the model group(P<0.05).The expression of HSL protein in curcumin group was significantly up-regulated(P<0.05).The protein expression of β3-AR was not significantly different in each group.The protein expression of C/EBPa and PPARy was significantly up-regulated in the treatment group(P<0.05).(P<0.05),while the curcumin group had no significant effect on the protein expression of PPARa in the metfonnin group and salvianolic acid group B significantly.Salbutamol group B significantly reduced the expression of SREBP-l protein(P<0.01),while the curcumin group and metformin group showed a decreasing trend,but there was no significant difference(P>0.05).Compared with the model group,the expression of ATGL and β3-AR in the curcumin group was significantly higher than that in the model group(P<0.05),and the expression of ATGL and P3-AR in the treatment group was significantly higher than that in the model group There was no significant difference.(P<0.05).Compared with the model group,the expression of PPARy protein was down-regulated in the treatment group(P<0.05).Although the protein expression of C/EBPa was down-regulated in each treatment group,(P>0.05).In addition,salbutamol group B and metfonnin group also significantly reduced the expression of SREBP-1 protein(P<0.01).Conclusions1.CTXKG blood circulation components,salicin can reduce the body fat and body f-at content of HFD-induced obese C57BL/6J mice,reduce the blood serum TG,TC,low density lipoprotein and FFA in obese mice,Density lipoprotein,and can reduce obesity-induced liver damage in mice,regulate lipid metabolism,improve lipid metabolism disorders.2.JTXKG blood circulation components Salvianolic acid B,curcumin can reduce obesity C57BL/6J mice FBG,increase glucose tolerance,improve insulin sensitivity,reduce obesity caused by IR,improve the IR state,regulate glucose metabolism,improve sugar Metabolic disorders.3.The effect of estrogenic acid B and curcumin on the expression of glycolipid metabolism in obese C57BL/6J mice may be involved in adipogenic differentiation by regulating transcription factors CEBPa,PPAR y and SREBP-1;HSL and β3-AR are involved in adipose splitting.Part Ⅱ The influence of blood circulation components in JTXKG on 3T3-L1 preadipocyte differentiation and functionObjectiveIn this part,we studied the differentiation of 3T3-L1 preadipocytes by studying the cell biological characteristics such as morphology,growth and lipid content of 3T3-L1 preadipocytes,and used JTXKG to promote blood stasis.The effects of JTXKG Blood circulation components on the expression of 3T3-L1 preadipocyte adipogenic differentiation factor(RT-PCR)were detected by RT-PCR and Western blot in the presence of two major monomers.And to explore the possible mechanism of JTXKG Blood circulation components on adipocyte differentiation and function.MethodsThe 3T3-L1 preadipocytes were cultured in DMEM high glucose medium containing 10%calf serum at 37℃,5%CO2 saturated humidity.When the cells were in good condition,they were inoculated on the culture plate.When the cells were covered with 90%or more,the cells were inoculated with 3T3-L1 cells(containing 850nM insulin,1μM DEX and 0.5mM IBMX)for 48h,-L1 cell differentiation solution(containing 850nM insulin medium)to continue to differentiate,every 48h to replace the cell differentiation fluid 1,8-12 days after differentiation of cells mature.(50μM,75μM,100μM);curcumin group(10μM,20μM,35μM)in the normal control group,divided into normal control group,metformin group(50μM),salvianolic acid group B Blank group was given equal volume of DMSO normal medium for homogeneous control,each group of four wells,drug intervention 48h.The levels of glycerol and glucose consumption were measured by colorimetric method.The differentiation and lipolysis of 3T3-L1 cells were observed after drug intervention.The cells and lipid droplets were observed by oil red O staining.RT-PCR was used to detect adipocytes The expression of ATGL,HSL,β3-AR,CEBP α,PPARα,PPARγ and SREBP-1 mRNA in adipogenic differentiation and adipogenic differentiation.ResultsThere was no significant difference in the inhibitory effect of salvianolic acid B at the concentration of 50,75 and 100 μM,and when the concentration was ≥125 u M,the effect of salvianolic acid B on the proliferation and viability of cell cells was significantly higher than that of the control group There was significant difference in cell growth inhibition(P<0.01).When curcumin concentration was ≥ 50 μ M,the inhibitory effect of curcunin oin cell growth was statistically significant(P<0.01).And the cells appeared to fall off and die.Salvianolic acid B,curcumin can promote the differentiation of 3T3-L1 preadipocytes and increase lipid accumulation.The concentration of 100μM saivianolic acid B to promote differentiation,increase the role of lipid accumulation of the strongest,showing a dose-effect relationship.The dose-effect relationship of curcumin at different concentrations was not significant.In the glucose consumption,50μM,75μM salvianolic acid significantly increased glucose consumption(P<0.05),and 100μM concentration of salvianolic acid B on glucose consumption is not obvious.(P<0.05),and the concentration of curcumin was the most significant(P<0.01)at 20μM and curcumin at 35μM.At the time of 48 h,the concentration of salvianolic acid B and curcumin could increase the glucose consumption(P<0.05),and 50μM,75μM salvianolic acid B and 10μM concentration of curcumin had the most significant effect on glucose consumption(P<0.01).The effect of salvianolic acid B on the release of glycerin from 3T3-L1 adipocytes was significantly lower than that of the control group(P<0.01)There was no significant difference with the blank group.At the time of 48 h,the levels of salvianolic acid and curcumin could significantly inhibit the glycerol release of 3T3-L1 adipocytes,the difference was statistically significant(P<0.01).(P<0.05).Compared with the control group,salvianolic acid B significantly reduced the mRNA expression of ATGL and HSL(P<0.05).The high concentration of lipopolysaccharide(P<0.05).The expression of PPARy mRNA was significantly up-regulated by salvianolic acid B(P<0.01).(P<0.05).The expression of PPARa mRNA was upregulated by high concentration of salvianolic acid.The expression of curcumin in 3T3-L1 adipocytes was significantly lower than that in the control group(P<0.05).Medium and high concentration of curcumin could significantly down-regulate the expression of HSL(P<0.01).High concentration of curcumin significantly down-regulated the mRNA expression of C/EBPa(P<0.05).The concentration of curcumin was significantly up-regulated by PPARy mRNA expression(P<0.05).The concentration of curcumin in each concentration group increased the expression of PPARamRNA,but there was no significant difference.Conclusions1.Salvianolic acid B,curcumin can promote 3T3-L1 preadipocyte differentiation,increase fat consumption and utilization of glucose,improve IR.2.Salvianolic acid B,curcumin through the promotion of differentiation and then enhance the fat cell function,inhibit the synthesis and decomposition of adipocytes TG,promote fatty acid peroxidation,reduce FFA release,regulate lipid metabolism,effective in weight loss lipid-lowering.3.Salvianolic acid B and curcumin on the differentiation and function of 3T3-L1 preadipocytes,which may be involved in adipogenic differentiation through salvianolic acid B and curcumin-regulated transcription factors CEBPa,PPARy,PPARa and SREBP-1;ATGL.HSL and β3 3-AR involved in lipolysis and effective. |
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